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Construction, production, and characterization of recombinant scFv antibodies against methamidophos expressed in Pichia pastoris

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Abstract

Pichia pastoris was used to express a recombinant scFv antibody against methamidophos derived from a recombinant phage-display library. The specific scFv gene was amplified from a positive clone and then subcloned into the expression vector pPICZα C. The resulting plasmid, pPICZα C–scFv, was linearized and transformed into P. pastoris (X-33). A transformant named X-33-Pp-Met-28D4, which showed strong expression of antibodies, was isolated, and the culture conditions were optimized. Under optimal conditions, P. pastoris cultures yielded much higher levels of scFv product than the Escherichia coli expression system. Immunochemical characterization of the scFv antibodies produced in P. pastoris indicated that the affinity and specificity of scFv against methamidophos are comparable to those of scFv antibodies produced in E. coli. Recoveries of methamidophos-fortified samples demonstrated that the P. pastoris-derived scFv antibodies can be used to determine the content of methamidophos residue in environmental and agricultural samples. For our purposes, expression in Pichia proved to be an efficient and economical method for the large-scale production of functional scFv antibodies against methamidophos for downstream applications.

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Acknowledgements

Financial support was from National “863” Project (2006AA10Z447) and the Department of Agriculture for Important Agricultural Technologies (2003-07-02A) of China.

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Authors and Affiliations

  1. Key Lab of Monitoring and Management of Plant Diseases and Pests, Ministry of Agriculture, College of Plant Protection, Nanjing Agricultural University, Nanjing, 210095, China

    Tiejun Li, Jianren Cheng, Baishi Hu, Yan Liu, Guoliang Qian & Fengquan Liu

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  1. Tiejun Li
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  2. Jianren Cheng
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  3. Baishi Hu
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  4. Yan Liu
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  5. Guoliang Qian
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  6. Fengquan Liu
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Correspondence to Fengquan Liu.

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Li, T., Cheng, J., Hu, B. et al. Construction, production, and characterization of recombinant scFv antibodies against methamidophos expressed in Pichia pastoris . World J Microbiol Biotechnol 24, 867–874 (2008). https://doi.org/10.1007/s11274-007-9554-9

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  • DOI: https://doi.org/10.1007/s11274-007-9554-9

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